Preliminary evidence indicates that muscle mitochondrial (mito) dysfunction in response to insulin occurs in T2D patients. Insulin in association with amino acids (AA) has an important regulatory role on muscle mito function. It remains to be determined whether muscle mito response to insulin is reduced in T2D patients because of insulin resistance or if insulin resistance causes mito dysfunction. To understand the underlying mechanism of mito dysfunction in T2 D patients we developed methods to simultaneously measure synthesis rates of multiple muscle mito proteins, transcript levels of genes encoding mito and transcription factors proteins and ATP production from needle muscle biopsy samples in human. The current proposal will determine whether muscle ATP production and protein synthesis is altered in T2D patients in comparison with non-diabetic people while on similar insulin levels. The underlying mechanism of this defect will be investigated by comparing transcript levels using custom gene arrays that we have developed, phosphorylation of signaling proteins and interaction of insulin and AA in regulating muscle mito protein synthesis and ATP production in T2D patients and non-diabetic people. Studies will be controlled with a somatostatin clamp of insulin, glucagon, and GH with varying dose of insulin and essential amino acid mixtures. We will further investigate whether pharmacological insulin sensitizers enhance muscle mito function in people with T2D. In order to clearly define the roles of insulin and hyperglycemia in regulating muscle mito functions, we will perform studies in T1D patients during insulin deficiency (hyperglycemia) and insulin replacement with high and normal glucose levels. Results from previous funding period also demonstrated that AA are predominant regulators of splanchnic protein synthesis and in vitro studies demonstrated selective effects of AA on gene transcripts and protein synthesis. We developed methods to simultaneously measure fractional synthesis rates of multiple plasma proteins (most of which originate from the liver). These proteins have critical functions including underlying roles in the pathophysiology of macrovascular disease. The proposed studies will examine the roles of insulin and AA and their interaction in regulating synthesis rates of plasma proteins with well-established functions. The proposed studies will for the first time provide information on the roles of AA and insulin in regulating synthesis of specific muscle mito and plasma proteins. The potential functional impact of changes in insulin and AA concentrations and insulin resistance on protein synthesis will be determined. In addition, the studies will hopefully provide novel insights on pathophysiology muscle insulin resistance and mito functions in T2D patients.